Sp1, one of the first gene-specific, metazoan transcription factors identified and cloned, is a ubiquitously expressed essential protein that regulates a variety of cellular and viral promoters (Dynan and Tjian, 1983, Cell 35:79-87; Jones and Tjian, 1985, Nature: 179-82; Kadonaga et al., 1987, Cell 51:1079-90; Marin et al., 1997, Cell 89:619-28; Saffer et al., 1991, Mol. Cell. Biol. 1:2189-99). Sp1 binds to DNA elements known as GC boxes via three Cys2His2 zinc finger domains, and interacts with the general transcription machinery through two glutamine-rich transactivation domains, designated A and B (FIG. 1) (Gidoni et al., 1984, Nature 312:409-13; Gidoni et al., 1985, Science 230:511-7; Hoey et al., 1993, Cell 72:247-60; Kadonaga et al., 1987, Cell 51:1079-90; Tanese et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:13611-6). The majority of TATA-less genes have multiple Sp1 sites in the proximal promoter region (Black et al., 1999, J. Biol. Chem. 274:1207-15), and more than half of expressed genes are TATA-less (Yang et al., 2000, FASEB J. 14:379-90). As such, Sp1 plays a global role in controlling gene expression.
Sp1 activity is significantly regulated through post-translational modifications, including phosphorylation, O-linked glycosylation, acetylation, and sumoylation (Jackson et al., 1990, Cell 63:155-65; Jackson and Tjian, 1988, Cell 55:125-33; Spengler and Brattain, 2006, J. Biol. Chem. 281:5567-74). The most studied modification is phosphorylation; Sp1 is phosphorylated by several kinases in vitro, including DNA-PK, casein kinase II, and cyclin A/cdk2, resulting in both positive and negative effects on transcription (Armstrong et al., 1997, J. Biol. Chem. 272:13489-95; Fojas de Borja et al., 2001, EMBO J. 20:5737-47; Jacksone et al., 1990, Cell 63:155-65 Ryu et al., 2003, J. Neuroscience 23:3597-606).
Several studies have also implicated Sp1 in the cellular response to DNA damage. In human cell lines exposed to ionizing radiation (IR), Sp1 DNA binding activity has been shown to increase in a transient and reversible manner (Meighan-Mantha et al., 1999, Mol. Cell. Biochem. 199:209-15; Yang et al., 2000, FASEB J. 14:379-90). Also, in cortical neurons, Sp1 DNA binding was shown to increase in response to oxidative stress, and Sp1 over-expression protected neurons from oxidative stress-induced cell death (Ryu et al., 2003, J. Neurosci. 23: 3597-606).
The human genome faces is continually threatened by DNA damage from reactive oxygen species (ROS) generated during aerobic respiration, cellular oxidase activity, and exposure to ionizing radiation (IR; Evans et al., 2004, Mutat. Res. 567:1-61). ROS-induced DNA damage includes small or bulky modifications to bases and sugars, inter- and intra-strand crosslinks, as well as single- and double-strand breaks (SSBs and DSBs, respectively) (Evans et al., 2004, Mutat. Res. 567:1-61; Roberfroid and Calderon, 1995, Free Radicals and Oxidation Phenomenon in Biological Systems. Marcel Dekker, Inc., New York). Molecular networks that rapidly sense and repair damage have evolved to maintain genomic stability and ensure cell survival.
Most threatening to genomic stability are DSBs, which activate the PI3 kinase-related kinases (PIKKs), including ATM (Ataxia-Telangiectasia Mutated), DNA-PK (DNA dependent protein kinase) and ATR (ATM and Rad3 related) (Abraham, 2004, DNA Repair 3:883-7). Cells deficient in PIKKs exhibit accumulated oxidative damage, radiation sensitivity, and impaired cell cycle checkpoint activation in response to DNA damage (Shiloh and Kastan, 2001, Adv. Cancer Res. 83:209-54). ATM protein, which is defective in the hereditary cancer-prone disorder Ataxia-Telangiectasia (A-T), is activated by DSBs and phosphorylates a variety of proteins involved in the DNA damage response leading to cell cycle checkpoint activation, DNA repair, altered gene expression patterns, and/or apoptosis (Shiloh, 2006, Trends Biochem. Sci. 31:402-10). Among the ATM substrates are several transcription factors, including p53 (Siliciano et al., Genes Dev. 11: 3471-81), BRCA-1 (Cortez et al., 1999, Science 286:1162-6), ATF2 (Bhoumik et al., 2001, Mol. Cell. 18:577-87), CREB (Shi et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101:5898-903), E2F1 (Lin et al., 2001, Genes Dev. 15:1833-44), and NF-κB regulators NEMO and IKK (Wu et al., 2006, Science, 311:1141-6). ATR, which is predominantly activated by bulky lesions and stalled replication forks, shares many substrates with ATM. The histone variant H2AX is phosphorylated by ATM, ATR and DNA-PK over a large region of chromatin surrounding a DSB (Rogakou et al., 1999, J. Cell. Biol. 146:905-16; Rogakou et al., 1998, J. Biol. Chem. 273: 5858-68).
Accumulated oxidative damage to genomic DNA is a recognized source of cancer; DSB are the single greatest threat to genomic stability whether caused by exposure to ROS generated during aerobic respiration, cellular oxidase activity, or exposure to ionizing radiation or chemotoxins. Methods of detecting DNA damage as a result of an individual's exposure to such agents are urgently needed, especially in the field of oncology. The present invention fulfills this need.